1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 April 2022 to 13 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The source of the inoculum of activated sludge was Sewage Treatment Plant, Eurofins Advinus Agrosciences Services India Private Limited, Bengaluru, India.
The inoculum was collected on 13 April 2022 at 08:45 AM from near the exit of the aeration tank of well operated wastewater treatment plant receiving predominantly domestic sewage. The sludge was allowed to settle for 15 minutes, and upper layer of finer solids was decanted. The collected sludge was centrifuged for 10 minutes at 3000 rpm and supernatant liquid was discarded. Then sludge was suspended in chlorine free tap water with shaking and re-centrifuge to remove wash-water and was discarded. Washing and centrifuging procedure was repeated three times.
A sample of washed sludge was weighed in triplicate and dried at 150°C in oven for 1 hour. From the obtained result the quantity of sludge required to obtain an activated sludge concentration of 3 g/L was calculated. During range finding test, 48.0601 g sludge was suspended in distilled water to make 6 L and 47.3400 g sludge was suspended in distilled water to make 6 L during definitive test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

21.4 - 21.8 °C

pH:

Refer to tables 1 and 2 in section "Any other information on results incl. tables"

Dissolved oxygen:

Refer to tables 1 and 2 in section "Any other information on results incl. tables"

Nominal and measured concentrations:

Nominal: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL beakers
- Aeration: forced aeration of 0.8-0.9 L/min to keep dissolved oxygen concentration above 60 to 70% saturation for 3 hours.
- No. of vessels per concentration (replicates): 3
- No. of vessels per blank control (replicates): 2
- No. of vessels per abiotic control (replicates): abiotic control was not included in the definitive test since there was no oxygen uptake at the highest test item concentration of 1000 mg/L during range finding test.
- Nutrients provided for bacteria: synthetic sewage feed as per OECD Guideline 209.
- Nitrification inhibitor used: N-allylthiourea, 3 replicates per concentration and 2 replicates per blank control.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled water containing <1 mg/mL TOC.

TEST CONCENTRATIONS
A range finding test was performed to determine the range of concentrations of test item to be used in the definitive test. The range finding test was performed with test item concentration at 10, 100 and 1000 mg/L along with a blank control and one abiotic control at 1000 mg/L test item concentration. For each group, one replicate was used.
Based on the results of the range finding test, the definitive test was carried out at the limit concentration of 1000 mg/L, with two sets of mixture to determine the total, heterotrophic and nitrification respiration inhibition. Since there was no oxygen uptake in the abiotic control at the highest test item concentration of 1000 mg/L during the range finding test, an abiotic control was not included in the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The pH, oxygen consumption rate and percent inhibition of total respiration and heterotrophic respirations are presented in tables 1 and 2 under "Any other information on results incl. tables".
The pH of the mixtures after 3 hours incubation ranged from 8.19 to 8.27.
No significant inhibition of AP 729 was observed in both total respiration and heterotrophic respiration. The nitrification respiration rate was found to be not significant (significance was –0.268%). Hence the inhibition of AP 729 due to nitrification respiration was not calculated.

Results with reference substance (positive control):

The reference substance 3,5-Dichlorophenol was tested at concentrations of 2, 8 and 32 mg/L.
The calculated percent inhibition for total respiration was 39.2, 61.7 and 95.1 % and the heterotrophic respiration was 23.5, 61.8 and 93.5 % at the tested concentrations, respectively.
The EC50 value of 3,5-Dichlorophenol for total respiration inhibition is 3.68 mg/L with fiducial limits at 95% ranging from 2.041 to 17.588 mg/L.
The EC50 value of 3,5-Dichlorophenol for heterotrophic respiration inhibition is 5.29 mg/L with fiducial limits at 95% ranging from 4.263 to 6.559 mg/L.

Reported statistics and error estimates:

The percentage inhibition of total respiration, heterotrophic respiration and nitrifying respiration was analyzed with the statistical method of Probit analysis.
The dose response curve was plotted and the slope of the curves and the median effective concentration (EC50) with 95% confidence limits as per maximum likelihood method of Finney (1978) was calculated using a validated computer program.

The pH, oxygen consumption rate and percent inhibition of total respiration and heterotrophic respirations of the definitive test are presented in the tables 1 and 2, respectively.

 

Table 1 - Total Respiration Inhibition 

Group No.	Description (mg/L)	pH of mixture at the end of incubation	Oxygen consumption rate (mg/L min)	Oxygen consumption rate (mg/L h)	Specific respiration rate ^a (mg/g h)	Percent inhibition, total respiration
G1	Blank control at start of test	8.20	0.5055	30.330	20.220	-
Test Item
G2	1000	8.27	0.5073	30.438	20.292	-0.7
G3	1000	8.26	0.5091	30.546	20.364	-1.1
G4	1000	8.25	0.5082	30.492	20.328	-0.9
Reference Item
G5	2	8.24	0.3064	18.384	12.256	39.2
G6	8	8.21	0.1927	11.562	7.708	61.7
G7	32	8.23	0.0245	1.470	0.980	95.1
G8	Blank control at end of test	8.25	0.5018	30.108	20.072	-

 

Table 2 - Heterotrophic Respiration Inhibition 

Group No.	Description (mg/L)	pH of mixture at the end of incubation	Oxygen consumption rate (mg/L min)	Oxygen consumption rate (mg/L h)	Specific respiration rate ^a (mg/g h)	Percent inhibition, heterotrophic respiration
G1N	Blank control at start of test	8.19	0.5045	30.270	20.18	-
Test Item
G2N	1000	8.20	0.5082	30.492	20.328	-0.6
G3N	1000	8.23	0.5055	30.330	20.220	-0.1
G4N	1000	8.25	0.5082	30.492	20.328	-0.6
Reference Item
G5N	2	8.27	0.3864	23.184	15.456	23.5
G6N	8	8.22	0.1927	11.562	7.708	61.8
G7N	32	8.24	0.0327	1.962	1.308	93.5
G8N	Blank control at end of test	8.26	0.5055	30.330	20.220	-

Note: ^a: oxygen consumed per g dry weight of sludge per hour.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 and NOEC values for total respiration inhibition and heterotrophic respiration inhibition of AP 729 are >1000 mg/L.

Executive summary:

The toxic effect of AP 729 on microorganisms (largely bacteria) from activated sludge of a well operated wastewater treatment plant receiving predominantly domestic sewage was studied according to OECD Guideline 209. 

A range finding test was performed to determine the range of concentrations of test item to be used in the definitive test. Based on the results of the range finding test, the tested concentration of AP 729 in the definitive test was at the limit concentration of 1000 mg/L in triplicate and in two sets, along with blank control (without test item) and reference substance (3,5-Dichlorophenol) at concentrations of 2, 8 and 32 mg/L. One set of all test groups was used to determine total respiration and another set of all test groups added with N-allylthiourea at 11.6 mg/L was used to determine the heterotrophic respiration and the nitrification potential of the sludge.

The test item mixtures and the reference substance mixtures were prepared by adding the required quantity of test item and reference substance directly into the test vessels to obtain different nominal concentrations, respectively.

Blank control, test mixtures and reference substance mixtures were aerated for the exposure time of 3 hours at a temperature between 21.4 and 21.8°C. The oxygen consumption rate in the mixture was measured for 10 minutes using dissolved oxygen electrode and the respiration rate was calculated.

No significant inhibition of AP 729 was observed in both total respiration and heterotrophic respiration.
Moreover, the nitrification respiration rate was found to be not significant: hence the inhibition of AP 729 due to nitrification respiration was not calculated.

The validity criteria of the test were fulfilled as:

• The blank controls oxygen uptake rate was 20.146 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour, which is more than the required 20 mg/g. The coefficient of variation of oxygen uptake rate in control replicates was 0.52 % which is within allowed 30% at the end of test.


• The EC₅₀ of 3,5-Dichlorophenol for total and heterotrophic respiration was 3.68 and 5.29 mg/L, which is in the range 2 to 25 mg/L for total respiration and 5 to 40 mg/L for heterotrophic respiration, respectively.

Therefore, the EC₅₀ and NOEC (No Observed Effect Concentration) value for total respiration inhibition and heterotrophic respiration inhibition of AP 729 are higher than 1000 mg/L.

Description of key information

The toxic effects of AP 729 on microorganisms from activated sludge of a well operated wastewater treatment plant receiving predominantly domestic sewage were studied according to OECD Guideline 209. 
The tested concentration of AP 729 was at the limit concentration of 1000 mg/L in triplicate and in two sets, along with blank control and reference substance (3,5-Dichlorophenol). One set of all test groups was used to determine total respiration and another set of all test groups added with N-allylthiourea at 11.6 mg/L was used to determine the heterotrophic respiration and the nitrification potential of the sludge.
Blank control, test mixtures and reference substance mixtures were aerated for the exposure time of 3 hours. No significant inhibition of AP 729 was observed in both total respiration and heterotrophic respiration.
Therefore, the EC₅₀ and NOEC value for total respiration inhibition and heterotrophic respiration inhibition of AP 729 are >1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L